Sensors for analyte detection and methods of manufacture thereof

ABSTRACT

Disclosed herein is a sensor comprising a conduit; the conduit comprising an organic polymer; a working electrode; the working electrode being etched and decorated with a nanostructured material; a reference electrode; and a counter electrode; the working electrode, the reference electrode and the counter electrode being disposed in the conduit; the working electrode, the reference electrode and the counter electrode being separated from each other by an electrically insulating material; and wherein a cross-sectional area of the conduit that comprises a section of the working electrode, a section of the reference electrode and a section of the counter electrode is exposed to detect analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application having Ser.No. 15/226,150 filed Aug. 2, 2016 which is a divisional of U.S. patentapplication having Ser. No. 13/164,656 filed on Jun. 20, 2011, now U.S.Pat. No. 9,402,574, which claims priority to U.S. Provisional PatentApplication No. 61/398,498 filed on Jun. 25, 2010, the entire contentsof which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

The present invention was developed in part with funding from the USArmy/TATRC under Grant # W81XWH-07-10668 and Grant # W81XWH-09-1-0711.The United States Government has certain rights in this invention.

BACKGROUND

This disclosure relates to sensors for analyte detection and to methodsof manufacture thereof. More specifically, this disclosure relates tobiosensors and to methods of detection of biological metabolites andother analytes.

Numerous clinical trials and intensive research efforts have indicatedthat continuous metabolic monitoring holds great potential to provide anearly indication of various body disorders and diseases. In view ofthis, the development of biosensors for the measurement of metaboliteshas become an area of significant scientific and technological study forvarious research groups across the world. A useful class of biosensorsare electrochemical sensors that link enzymatic reactions toelectroactive products. These sensors also enable the detection of smallvolumes of bio-analytes in clinical or home use applications. Forexample, the development of miniaturized implantable sensors forcontinuous monitoring of glucose is useful for optimal care of diabetesmellitus. Many other clinical situations also necessitate themeasurement of various body metabolites like lactate, creatinine,creatine, glutamate, phosphate, cysteine, homocysteine, and the like.For example, a device that can measure lactate levels has importantimplications in a number of diseases and conditions (e.g., to indicatemuscle fatigue, shock, sepsis, kidney disorders, liver disorders andcongested heart failure). In some clinical situations, simultaneousmonitoring of two or more metabolites is desirable.

For example, the complex interrelationship between glucose and othermetabolic analytes induces one to simultaneously detect glucose,glutamate, lactate, oxygen, carbon dioxide, and the like. Simultaneousmonitoring of brain glucose, lactate and oxygen gives a comprehensivepicture of complementary energy supply to the brain in response to acuteneuronal activation. Levels of glucose and glutamate in cerebrospinalfluid are important in the control of diseases such as meningitis.

Currently, most of the electrochemical sensors used for the specificdetection of lactate, glucose, glutamate, and the like, employ analytespecific enzymes, and are based on the Clark-type amperometricdetection. For example, first generation Clark-type glucose sensorsemploy the glucose oxidase enzyme (GOx), immobilized on top of a workingelectrode. This enzyme catalyses the oxidation of glucose toglucarolactone, as shown in reaction (1).

The generated H₂O₂ is amperometrically assessed on the surface of aworking electrode according to reaction (2) shown below.

Currently, these biosensors suffer from two major pitfalls: (i) lack ofminiaturization compatible with roll-to-roll production and (ii) lack ofhigh sensor performance that is desirable for most of the in vivoapplications.

The desire for miniaturization occurs from the complex applicationsthese biosensors are being utilized for. Typical applications for thesein vivo biosensors include metabolite monitoring in the neurons, extracellular space, eyes, subcutaneous (s.c.) tissue, veins, and the like.In some cases, the biosensor is used in the affected area of theparticular organ to diagnose the ailment and is left in place to monitorthe condition of the ailment. In all of these applications, it isdesirable to have miniaturization in order to avoid damage to thehealthy tissue and to reduce wound recovery time, infection and patientdiscomfort.

Miniaturization of biosensors and sensor performance are twodiametrically opposed issues. For example, most of the currentminiaturization strategies result in a decrease in sensor performance asa result of reduced active working area, reduced enzyme loading andreduced signal-to-noise ratio. Any increase in the size of the biosensorwill cause a large damage to the local tissue that will augment themagnitude of a foreign body response. This foreign body response willfurther decrease sensor performance by decreasing the analyte flux andpossibly denaturing the sensing enzymes.

To alleviate some of the aforementioned issues of miniaturization, anumber of micro-sensory devices based on microelectromechanical systems(MEMS) technology have been reported with advantages such as highprecision, high functionality and mass-production. However, thistechnology uses expensive equipment and materials that could lead to anoverall increase in the cost per piece of the biosensor. Moreover, thesedevices are based on inflexible or brittle materials such as silicon andglass, which have a higher chance of breakage within the in vivoenvironment.

In order to simultaneously afford miniaturization while at the same timeincreasing enzyme loading (to improve sensor performance), a siliconmicro-machined needle-shaped structure for glucose monitoring has beenreported. These needle-shaped biosensors along with channels for fluidflow and enzyme housing are created by wet and dry etching processes,while the (Ti/Pt) titanium/platinum working and (Ag/AgCl) silver/silverchloride reference electrodes located at the tip of the needle-shapedbiosensors are patterned by photolithography. However as mentionedabove, since these devices are made up of silicon substrates, these aremore prone to breakage within the body.

In order to avoid the problem of sensor breakage in the body, biosensorshave been fabricated, where the sensing electrodes are patterned on apolymeric KAPTON® film and subsequently rolled up to form a twodimensional cylindrical electrode. While the soft and flexible nature ofthese sensors presents an advantage over currently available techniques,these sensors are not reproducible on large scale and have a largesensor-to-sensor variability because of the large effect of the roll-upangle on the performance of the sensor. Moreover, the problem of enzymeloading and low electroactive surface still persists.

Biosensor miniaturization based on electrodes patterned on planar (bothrigid as well as flexible) substrates have also been reported. However,these planar sensors do not afford 3-D analyte diffusion that isdesirable for enhanced sensor performance. For enhanced sensorperformance, miniaturized sensors based on micro-disc array electrodeshave been reported, which use expensive machinery and cannot be easilyproduced at lower cost. Furthermore, these have problems of lowelectroactive area, lack of reusable electrodes and reduced enzymeloading.

Various reports have also emerged on the use of electrodes decoratedwith nanostructured materials such as nanoparticles, nanotubes andnanocubes to enhance the electroactive areas and enzyme loading. Thesenanostructured materials tend to be fouled very quick, thereby resultingin a quick loss of sensor performance.

Based on the above, it is desirable to develop methodologies for theproduction of biosensors that simultaneously afford extrememiniaturization, high sensor performance and flexibility forroll-to-roll production. Such methodologies will improve the quality ofpoint of care diagnosis and prognosis of various body disorders.

SUMMARY

Disclosed herein is a sensor comprising a conduit; the conduitcomprising an organic polymer; a working electrode; the workingelectrode being etched and decorated with a nanostructured material; areference electrode; and a counter electrode; the working electrode, thereference electrode and the counter electrode being disposed in theconduit; the working electrode, the reference electrode and the counterelectrode being separated from each other by an electrically insulatingmaterial; and wherein a cross-sectional area of the conduit thatcomprises a section of the working electrode, a section of the referenceelectrode and a section of the counter electrode is exposed to detectanalytes; and wherein an old section of the sensor can be severed off toexpose a new section of the sensor that can be used to detect analytes.

Disclosed herein too is a sensor comprising a first tape; a second tape;the second tape being opposedly disposed on the first tape and incontact with the first tape; a working electrode, a reference electrodeand a counter electrode, each being disposed between the first tape andthe second tape; the working electrode, the reference electrode and thecounter electrode not being in contact with one another; and wherein across-sectional area of the sensor that comprises a section of theworking electrode, a section of the reference electrode and a section ofthe counter electrode is exposed to detect analytes; and wherein an oldsection of the sensor can be severed off to expose a new section of thesensor.

Disclosed herein too is a method comprising disposing in a conduit, aworking electrode; a reference electrode; and a counter electrode; theworking electrode, the reference electrode and the counter electrodebeing separated from each other by an electrically insulating material;cutting the conduit to expose a section of the working electrode; asection of the reference electrode and a section of the counterelectrode; etching the exposed section of the working electrode; anddecorating the exposed section of the working electrode.

Disclosed herein too is a method comprising disposing a workingelectrode, a reference electrode and a counter electrode between a firsttape and a second tape; wherein the working electrode, the referenceelectrode and the counter electrode are not in contact with one another;cutting the first tape and the second tape to expose a cross-sectionalsurface area of the working electrode, the reference electrode and thecounter electrode; etching the exposed surface of the working electrode;and decorating the etched surface of the working electrode.

Disclosed herein too is a method comprising disposing a plurality ofthin wires within a conduit; the thin wires being electrically separatedby an insulating layer; cutting the conduit into a thin slice; polishingboth sides of the conduit to expose a cross-sectional area of the thinwire; electrochemically etching the exposed cross-sectional area of thethin wire; coating the exposed cross-sectional area withelectro-catalytic moieties suitable for the catalysis of a given redoxspecies; connecting a cross-sectional are of the thin wire that isopposed to the etched cross-sectional area to an active matrix that isin turn connected to an appropriate microelectronic device for signaldetection; where the active matrix switches and interrogates differentthin wires.

Disclosed herein too is a sensor comprising a sensor housing; an activematrix addressing chip; a plurality of electrodes disposed within anelectrically insulating casing; where each electrode has across-sectional area that is exposed to ambient surroundings; andwherein an opposing cross-sectional area of each of the electrodescontacts the active matrix addressing chip; and wherein the activematrix addressing chip and the plurality of electrodes being disposed ina sensor housing.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of a biosensor configuration whenfabricated in a tubular structure;

FIG. 1B is a scanning electron micrograph image of the etched Au wireedge;

FIG. 1C is a scanning electron image of the etched Au wire edge andsubsequently Platinum-decorated etched Au wire edge;

FIG. 2A is a schematic representation of a biosensor configuration of asensor, fabricated on a KAPTON® tape or similar flexible ‘tape’ likematerials. It reflects a first tape which forms a first side of thesensor;

FIG. 2B is a schematic representation of a biosensor configuration of asensor, fabricated on a KAPTON® tape or similar flexible ‘tape’ likematerials. In this figure, at least three electrically conductive wires(e.g., in the form of lines) are disposed on the tape;

FIG. 2C is a schematic representation of a biosensor configuration of asensor, fabricated on a KAPTON® tape or similar flexible ‘tape’ likematerials. This figure is a depiction of the sensor that contains thefirst tape, the opposing second tape and the wires;

FIG. 2D is a cross-section of the sensor taken from the FIG. 2C. In theFIG. 2D, the working electrode, the reference electrode and the counterelectrode are disposed side-by-side between the first tape and thesecond tape.

FIG. 3 is a response current as function of etching time during thepitting/etching process. Here, the bias is switched between +0.8 V and−1 V, with progressive increase in the response current due to thegradual increase in the surface area of the working electrode;

FIG. 4 depicts the sensitivity to H₂O₂ of a 25 micron wire-edgebiosensor of FIG. 1A at its various stages of fabrication;

FIG. 5 depicts the sensitivity of the biosensor configuration to H₂O₂before and after subjecting to an electrochemical cleaning procedure;

FIG. 6 is a graph showing a cyclic voltammogram in 10 mM K₃(FeCN₆) in 1MKCl aqueous solution at 10 mV/s scanning rate of the biosensorconfiguration of FIG. 1A at various stages of it fabrication. Thesigmoidal shapes of these curves indicate microelectrode behavior whichin turn demonstrates 3-D analyte diffusion;

FIG. 7A is a graph showing amperometric response of working electrodeswhen coated with PPD and PPD+FMN-NT operated at 0.7 V versus Ag/AgClreference for sequential additions of (a) 2 μM (b) 20 μM (c) 200 μM (d)2000 μM of H₂O₂ as represented by downward arrows;

FIG. 7B is a graph showing saturation amperometric current versus H₂O₂concentration for PPD and PPD+FMN-NT coated working electrode;

FIG. 8 depicts the configuration for obtaining a stable sensor structurein a planar configuration;

FIG. 9A is a schematic representation of various steps involved inmasking methodology that is used to fabricate a multi-analyte sensor;

FIG. 9B shows the response of the multi-analyte (glucose, lactate andoxygen) sensor to changes in lactate and glucose concentrations; and

FIG. 10 is a schematic configuration of interfacing a multi-wire sensorgeometry to an active matrix addressable chip located within aprotective encasing. This will allow addressing individual wire edgeplane sensors with minimum interconnects.

DETAILED DESCRIPTION

It will be understood that when an element is referred to as being “on”another element, it can be directly on the other element or interveningelements may be present therebetween. In contrast, when an element isreferred to as being “directly on” another element, there are nointervening elements present. As used herein, the term “and/or” includesany and all combinations of one or more of the associated listed items.

It will be understood that, although the terms first, second, third,etc. may be used herein to describe various elements, components,regions, layers and/or sections, these elements, components, regions,layers and/or sections should not be limited by these terms. These termsare only used to distinguish one element, component, region, layer orsection from another element, component, region, layer or section. Thus,a first element, component, region, layer or section discussed belowcould be termed a second element, component, region, layer or sectionwithout departing from the teachings of the present invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. It willbe further understood that the terms “comprises” and/or “comprising,” or“includes” and/or “including” when used in this specification, specifythe presence of stated features, regions, integers, steps, operations,elements, and/or components, but do not preclude the presence oraddition of one or more other features, regions, integers, steps,operations, elements, components, and/or groups thereof.

Furthermore, relative terms, such as “lower” or “bottom” and “upper” or“top,” may be used herein to describe one element's relationship toanother element as illustrated in the Figures. It will be understoodthat relative terms are intended to encompass different orientations ofthe device in addition to the orientation depicted in the figures. Forexample, if the device in one of the figures is turned over, elementsdescribed as being on the “lower” side of other elements would then beoriented on “upper” sides of the other elements. The exemplary term“lower,” can therefore, encompasses both an orientation of “lower” and“upper,” depending on the particular orientation of the figure.Similarly, if the device in one of the figures is turned over, elementsdescribed as “below” or “beneath” other elements would then be oriented“above” the other elements. The exemplary terms “below” or “beneath”can, therefore, encompass both an orientation of above and below.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

Exemplary embodiments are described herein with reference to crosssection illustrations that are schematic illustrations of idealizedembodiments. As such, variations from the shapes of the illustrations asa result, for example, of manufacturing techniques and/or tolerances,are to be expected. Thus, embodiments described herein should not beconstrued as limited to the particular shapes of regions as illustratedherein but are to include deviations in shapes that result, for example,from manufacturing. For example, a region illustrated or described asflat may, typically, have rough and/or nonlinear features. Moreover,sharp angles that are illustrated may be rounded. Thus, the regionsillustrated in the figures are schematic in nature and their shapes arenot intended to illustrate the precise shape of a region and are notintended to limit the scope of the present claims.

The transition term “comprising” encompasses the transition terms“consisting of” and “consisting essentially of.”

The term “flexible” as used herein is used to indicate that the devicecan be configured to take any desired position and can remain in thisposition for a desirable period of time. The device can return to itsoriginal position upon the application of a stimulus.

Various numerical ranges are disclosed herein. These ranges areinclusive of the endpoints as well as numerical values between theseendpoints. The numbers in these ranges and those on the endpoints areinterchangeable.

Disclosed herein is a flexible, miniature, reusable sensor that can beused for the detection of analytes and other biological metabolites. Thesensor can be placed in-vivo and will have minimum invasive effect onthe body of a living being. Disclosed herein too is a method ofmanufacturing the sensor. The sensor comprises a plurality of electrodesdisposed in a flexible conduit. In an exemplary embodiment, the sensorcomprises at least three electrodes—a first electrode or a workingelectrode, a second electrode or a counter electrode and a thirdelectrode or a reference electrode disposed in the flexible conduit andcontacted on their respective peripheries by the conduit. In avariation, the reference and the counter electrode could be the sameelectrode.

The sensor configuration utilizes the ‘edge’ or a “cross sectional area”or “cross sectional area surface” of an inert metal wire (such asplatinum, gold, silver, palladium, and the like) as a working electrode.This metal wire is placed next to two other metal wires each of whichwill serve as a reference and a counter electrode respectively. Thethree metal wires are placed in an ultra thin, flexible tubing and fixedin place with a polymer conduit. One end of this conduit is cut alongits short axis to expose the edges of the three metal wires, thesurfaces of which will serve as the active areas of working, referenceand counter electrodes. This configuration affords sensor regeneration,in that a new surface of these three electrodes can be readily obtainedby cutting the tubing at a different place.

The method for manufacturing the sensor comprises disposing a conduit onthe three electrodes. The conduit can be extruded onto the threeelectrodes or alternatively the three electrodes can be coated with amonomeric solution which is then cured to form the polymeric conduit.The sensor, which comprises the conduit with the three electrodes isthen cut at an angle that is perpendicular to a longitudinal axis of theconduit. The working electrode is then etched and decorated withnanostructured materials. The etching and nanostructuring of the workingelectrode makes the working electrode highly sensitive, therebyimproving the sensitivity and limit of detection of the sensor. Theworking electrode is also periodically cleaned in order to renew itsactivity.

In one method of using the sensor, the working electrode is coated withan enzyme or a plurality of enzymes, which will interact with an analyteof choice to produce an electroactive species. By changing the potentialacross the electrodes, the concentration of the electroactive speciescan be sensed and determined.

FIG. 1A is a schematic depiction of a cross-section of the sensor 100that comprises a conduit 102 in which are disposed three wires whichserve as the working electrode 104, the reference electrode 106 and thecounter electrode 108 respectively.

The conduit may have a cross-sectional area that is circular,rectangular, square, triangular, polygonal, ellipsoidal, or acombination comprising at least one of the foregoing shapes. Thecross-sectional area may be specially fabricated to conform to aparticular cavity of space available in the body of a living being.

It is desirable for the conduit to be flexible so that it can be easilybent by hand. In one embodiment, the conduit has an elastic modulus lessthan or equal to about 10⁶ Pascals, specifically less than or equal toabut 10⁵ Pascals. In another embodiment, the sensor has an elasticmodulus less than or equal to about 10⁶ Pascals, specifically less thanor equal to abut 10⁵ Pascals.

The conduit generally comprises an organic polymer. Organic polymersinclude a wide variety of thermoplastic polymers, blend of thermoplasticpolymers, thermosetting polymers, or blends of thermoplastic polymerswith thermosetting polymers. The organic polymer may also be a blend ofpolymers, copolymers, terpolymers, or combinations comprising at leastone of the foregoing organic polymers. The organic polymer can also bean oligomer, a homopolymer, a copolymer, a block copolymer, analternating block copolymer, a random polymer, a random copolymer, arandom block copolymer, a graft copolymer, a star block copolymer, adendrimer, a polyelectrolyte (polymers that have some repeat groups thatcomprise electrolytes), a polyampholyte (a polyelectrolyte having bothcationic and anionic repeat groups), an ionomer, or the like, or acombination comprising at last one of the foregoing organic polymers.

It is desirable for the organic polymer to be an elastomer at the bodytemperature of the living being into whom the sensor is inserted. In oneembodiment, the organic polymer is a shape memory polymer that canreturn to a pre-determined shape at the body temperature of the livingbeing into whom the sensor is inserted.

Examples of the organic polymers are polyacetals, polyolefins,polyacrylics, polycarbonates, polystyrenes, polyesters, polyamides,polyamideimides, polyarylates, polyarylsulfones, polyacrylates,polymethacrylates, polyethersulfones, polyphenylene sulfides, polyvinylchlorides, polysulfones, polyimides, polyetherimides, fluoropolymers,polyfluoroethylenes, polyetherketones, polyether etherketones, polyetherketone ketones, polybenzoxazoles, polyphthalides, polyacetals,polyanhydrides, polyvinyl ethers, polyvinyl thioethers, polyvinylalcohols, polyvinyl ketones, polyvinyl halides, polyvinyl nitriles,polyvinyl esters, polysulfonates, polysulfides, polythioesters,polysulfones, polysulfonamides, polyureas, polyphosphazenes,polysilazanes, styrene acrylonitrile, acrylonitrile-butadiene-styrene(ABS), polyethylene terephthalate, polybutylene terephthalate,polyurethane, ethylene propylene diene rubber (EPR),polytetrafluoroethylene, perfluoroelastomers, fluorinated ethylenepropylene, perfluoroalkoxyethylene, polychlorotrifluoroethylene,polyvinylidene fluoride, polysiloxanes, or the like, or a combinationcomprising at least one of the foregoing organic polymers.

Examples of polyelectrolytes are polystyrene sulfonic acid, polyacrylicacid, pectin, carageenan, alginates, carboxymethylcellulose,polyvinylpyrrolidone, or the like, or a combination comprising at leastone of the foregoing polyelectrolytes.

Examples of thermosetting polymers include epoxy polymers, unsaturatedpolyester polymers, polyimide polymers, bismaleimide polymers,bismaleimide triazine polymers, cyanate ester polymers, vinyl polymers,benzoxazine polymers, benzocyclobutene polymers, acrylics, alkyds,phenol-formaldehyde polymers, novolacs, resoles, melamine-formaldehydepolymers, urea-formaldehyde polymers, hydroxymethylfurans, isocyanates,diallyl phthalate, triallyl cyanurate, triallyl isocyanurate,unsaturated polyesterimides, or the like, or a combination comprising atleast one of the foregoing thermosetting polymers.

Examples of blends of thermoplastic polymers includeacrylonitrile-butadiene-styrene/nylon,polycarbonate/acrylonitrile-butadiene-styrene, acrylonitrile butadienestyrene/polyvinyl chloride, polyphenylene ether/polystyrene,polyphenylene ether/nylon, polysulfone/acrylonitrile-butadiene-styrene,polycarbonate/thermoplastic urethane, polycarbonate/polyethyleneterephthalate, polycarbonate/polybutylene terephthalate, thermoplasticelastomer alloys, nylon/elastomers, polyester/elastomers, polyethyleneterephthalate/polybutylene terephthalate, acetal/elastomer,styrene-maleicanhydride/acrylonitrile-butadiene-styrene, polyetheretherketone/polyethersulfone, polyether etherketone/polyetherimidepolyethylene/nylon, polyethylene/polyacetal, or the like.

In one embodiment, biodegradable polymers can also be used tomanufacture the conduit. Suitable examples of biodegradable polymers areas polylactic-glycolic acid (PLGA), poly-caprolactone (PCL), copolymersof polylactic-glycolic acid and poly-caprolactone (PCL-PLGA copolymer),polyhydroxy-butyrate-valerate (PHBV), polyorthoester (POE), polyethyleneoxide-butylene terephthalate (PEO-PBTP), poly-D,L-lacticacid-p-dioxanone-polyethylene glycol block copolymer (PLA-DX-PEG), orthe like, or combinations comprising at least one of the foregoingbiodegradable polymers. The biodegradable polymers upon undergoingdegradation can be consumed by the body without any undesirable sideeffects.

In one embodiment, an exemplary polymer is a thermosetting polymers. Anexample of a suitable elastomeric thermosetting polymer for use in theconduit is polydimethylsiloxane. In another embodiment, an exemplarypolymer is a fluoropolymers. An example of a suitable thermoplasticpolymer for use in the conduit is polytetrafluoroethylene. In yetanother embodiment, an exemplary polymer is a biodegradable polymer. Anexample of a suitable biodegradable polymer for use in the conduit ispolylactic-glycolic acid (PLGA).

The conduit has a diameter effective to embed and to surround the threeelectrodes with sufficient space between them to prevent any electricalinterference that may distort a signal. In one embodiment, the conduitis in intimate contact with the circumferential periphery of each of theelectrodes.

As detailed above, the working electrode, the reference electrode andthe counter electrode are each embedded in the conduit. The workingelectrode, the reference electrode and the working electrode eachcomprises an electrically conducting material such as for example ametal, a ceramic or a conductive polymer. Other electrically conductingmaterials can also be used to form each of the electrodes.

An exemplary electrically conducting material is a metal. Exemplarymetals are platinum group metals or noble metals. Examples of suitablemetals are platinum, gold, silver, palladium, rhodium, ruthenium,iridium, or the like, or a combination comprising at least one of theforegoing metals.

Examples of suitable electrically conducting ceramics are indium tinoxide, indium zinc oxide, antimony oxide, zinc oxide, or the like, or acombination comprising at least one of the foregoing ceramics.

Examples of suitable electrically conducting polymers are polyaniline,polypyrrole, polyacetylene, polythiophene, or the like, or a combinationcomprising at least one of the foregoing electrically conductingpolymers.

Examples of other conducting materials that can be used as the workingelectrode, the reference electrode or the counter electrode are carbonfibers, carbon nanotubes (e.g., single wall carbon nanotubes, doublecarbon wall nanotubes, multiwall carbon nanotubes), metal coated fibers,or the like, or a combination comprising at least one of the foregoingfibers. Exemplary wires comprise gold, platinum or carbon fibers.

In one embodiment, each of the electrodes is manufactured from abiodegradable electrically conducting material. An example of abiodegradable electrically conducting material is one of the foregoingbiodegradable polymers blended with an electrically conducting materialsuch as carbon black, carbon nanotubes, intrinsically electricallyconducting polymers (e.g., polypyrrole, polythiophene, polyaniline,polyacetylene, and the like).

The working electrode, the reference electrode and the counter electrodecan each be in the form of a wire. The wire can be a single wire or cancomprise multiple strands. In one embodiment, when multiple strands areused, the multiple strands are braided to form the wire, which serves asan electrode.

In one embodiment, the wire may be in the form of a ribbon. The wire hasa circular cross-section while the ribbon has a rectangularcross-section. The wire generally has a diameter of about 5 nanometersto about 1000 micrometers, specifically about 100 nanometers to about500 micrometers, and more specifically about 200 nanometers to about 25micrometers. If the wire is in the form of a ribbon, the ribbon willhave a width of a diameter of about 5 nanometers to about 100micrometers, specifically about 10 nanometers to about 50 micrometers,and more specifically about 20 nanometers to about 25 micrometers.

The disposing of the conduit on the electrodes can be accomplished byextrusion. Cross-head extrusion is generally used to dispose the conduiton the electrodes. The extrusion process is generally conducted withthermoplastic or thermosetting polymers. In an exemplary embodiment,thermoplastic polymers are used in the extrusion process to manufacturethe conduit.

In another embodiment, the conduit is disposed on the electrodes bymolding. Thermoplastic polymers and/or thermosetting polymers may beused to manufacture the conduit in a molding process.

Alternatively, the electrodes may be coated with a common coating of amonomeric material. The monomeric material is then crosslinked to form apolymer. Crosslinking can be accomplished by ultraviolet (UV) curing,thermally induced crosslinking, infrared induced crosslinking, X-rayinduced crosslinking, electron beam induced crosslinking, or acombination thereof. Thermosetting polymers result from the crosslinkingof the monomers. Examples of thermosetting polymers are provided above.

In one embodiment, in a variation of the aforementioned configuration,the metal wires and the flexible conduit are replaced by threeelectrodeposited metal conducting lines, sandwiched between two piecesof a KAPTON® (polyimide) tape, Scotch Tape or any other ultra-thinflexible polymer with an adhesive backing. In this configuration, thesurfaces of the three working electrodes are realized by cutting the‘tape sandwich’ across the shorter edge (at an angle between 10 and 170degrees and preferably between 45 and 90 degrees with respect to aperpendicular to the longer edge of the tape). The working electrode isfurther processed according to the aforementioned procedures detailedbelow to enhance its electro-active area and thereby its performance.

FIGS. 2A-2C are a depiction of the elements and the method used in themanufacturing of such a sensor. The FIG. 2A reflects a first tape whichforms a first side of the sensor. In the FIG. 2B, at least threeelectrically conductive wires (e.g., in the form of lines) are disposedon the tape. The wires may be disposed on the tape by electrochemicaldeposition or by chemical vapor deposition using a mask to prevent metaldeposition on those areas of the tape where deposition is not desired.

Electrochemical deposition involves exposing the tape to a metalcontaining solution and application of a constant potential or current.The application of constant potential or current includes techniquessuch as galvanometry, amperometry, potentiometry, cyclic voltammetry andthe like. If combinations of the foregoing electrochemical depositiontechniques are used, they may be employed simultaneously orsequentially.

Chemical vapor deposition includes atmospheric chemical vapordeposition, low pressure chemical vapor deposition, ultrahigh vacuumchemical vapor deposition, aerosol assisted vapor deposition, directliquid injection chemical vapor deposition, microwave plasma assistedchemical vapor deposition, remove plasma enhanced chemical vapordeposition, atomic layer chemical vapor deposition, hot wire (hotfilament) chemical vapor deposition, metal organic chemical vapordeposition, combustion chemical vapor deposition, vapor phase epitaxy,rapid thermal chemical vapor deposition, hybrid physical chemical vapordeposition, or a combination comprising at least one of the foregoingprocesses. If combinations of the foregoing chemical vapor depositionprocesses are used, they may be employed simultaneously or sequentially.

In case of use of electrochemical deposition for the disposition of thewires on the first tape, a thin layer of another organic film can beimmediately deposited (using chemical vapor deposition) around the edgesof the wires in order to increase their adhesion to the tape. Someexamples of this organic thin film include hexamethyldisilazane,hexamethyldisiloxane, trichloromethylchlorosilanes, hexachlorodisiloxane, tetramethyl(dichloromethyl)disiloxane and the like. Othermaterials such as the thermosetting or thermoplastic polymers (listedabove) may also be used to form the thin layer of organic film.

Following deposition of wires, a second tape may be disposed on thewires. The second tape is opposedly disposed to the first tape. Thesecond tape forms a second side of the sensor. The FIG. 2C is adepiction of the sensor that contains the first tape, the opposingsecond tape and the wires. Following the disposing of the second tape,the surface of one of the wires is etched and decorated with ananostructured material to form the working electrode. The etching andthe decorating of the working electrode are described in detail below.The disposing of the second tape, the etching and decorating and thecutting to expose an edge can be conducted in any desirable sequence.For example, the electrodes may be cut and decorated prior to disposingthe second tape on the first tape and on the wires.

FIG. 2D is a cross-section of the sensor 200 taken from the FIG. 2C. Inthe FIG. 2D, the working electrode 206, the reference electrode 208 andthe counter electrode 210 are disposed side-by-side between the firsttape 202 and the second tape 204. In one embodiment, the first tape andthe second tape contact each other via an adhesive. In anotherembodiment, the first tape and the second tape contact each other via anenzyme layer.

While the FIG. 2D depicts the working electrode, the reference electrodeand the counter electrode arranged in order from left to right, thepositions can be interchanged depending upon convenience. In addition,there can be more than three electrodes disposed upon the tape.

Following the disposing of the conduit on the electrodes, the conduitalong with the wires is cut at an angle that is not perpendicular to thelongitudinal axis of the conduit. The longitudinal axis of the conduitis one which is parallel to the circumferential surface of the conduit(i.e., it is concentric with the curved surface of the conduit). Thecutting of the conduit generates an edge in the working electrode thatcan be used for detection, thus producing an edge-based sensor. Thefollowing procedures are then performed on the conduit to improve theperformance of the sensor.

In one embodiment, the working electrode is selectively etched anddecorated with nanostructured particles to enhance the sensitivity ofthe sensor.

The surface of the working electrode is selectively etched to enhancethe electroactive area and is further decorated with highly activatednanostructured materials to enhance its activity towards electrochemicaloxidation or reduction. FIG. 1B is a scanning electron micrograph imageof an etched gold wire cross sectional surface.

Suitable etching agents can be either acidic or basic. Etching can beconducted simultaneously with an acid and then with a base or viceversa.

Examples of suitable acids for etching are hydrochloric acid, nitricacid, sulfuric acid, or the like, or a combination comprising at leastone of the foregoing acids. Examples of suitable bases for etching thewires are potassium hydroxide, sodium hydroxide, ammonium hydroxide,sodium hydrogen phosphate (Na₂HPO₄) or the like, or a combinationcomprising at least one of the foregoing bases. Neutral solutions canalso be used for etching. Exemplary solutions are basic (alkaline)solutions.

In one embodiment, directed to the pitting and etching process, apotential is applied between the working and the reference electrode toaccelerate the pitting and etching process.

In yet another embodiment, the ‘pitted and/or etched’ working electrodeis decorated with nanostructured materials by exposing the workingelectrode to a solution of the nanostructured materials and by applyinga constant potential or current and preferably potential between theworking and the reference electrodes. The nanostructured material couldbe a nanoparticle, nanotube or a nanocube. These nanostructuredmaterials can comprise platinum, silver, iridium, rhodium, gold,palladium, carbon, graphene, diamond, or the like, or a combinationthereof. In an exemplary embodiment, the nanostructured material iscarbon or platinum. FIG. 1C is a scanning electron image of the etchedgold wire (electrode) edge that is subsequently decorated with platinum.The term or prefix “nano” encompasses structures that have averageparticle sizes of less than or equal to about 200 nanometers,specifically less than or equal to about 100 nanometers.

In a variation of the aforementioned process, the nanostructuredmaterial can be co-deposited with a polymeric membrane by exposing theworking electrode to a solution contacting both the nanostructuredmaterials and a monomer and by applying a constant potential or currentand preferably potential between the working and the referenceelectrode. The concentration of the monomer can be varied and themonomer can be can be selected from the group consisting ofortho-phenylene diamine (OPD), para-phenylene diamine, meta-phenylenediamine, phenol, pyrrole, flavins, naphthalene, aniline, thiophenes,sulfonated aniline, sulfonated pyrrole, or the like, or a combinationthereof.

In a variation of the aforementioned process, the nanostructuredmaterial is coated with the monomer and then co-deposited on the surfaceof the working electrode, by applying a potential or a current betweenthe working and the reference electrode.

In another embodiment, the working electrode is coated with a specificset of enzymes, at least one of which will initiate a reaction with ananalyte of interest to produce an electroactive species. The enzymesselected from the group consisting of transferases, hydrolases,oxidases, peroxidases, kinases, superoxidases, phosphatases, or thelike. Enzyme deposition can be achieved by methods such as drop casting,dip coating, spin coating, spray coating and electrodeposition. In anexemplary embodiment, the enzyme deposition can be accomplished viaelectrodeposition. This electrodeposition can be achieved by applying aconstant current or a potential across the working and the referenceelectrodes. In an exemplary embodiment, the electrodepositon is achievedby applying a constant potential across the working and the referenceelectrodes.

In a variation of the aforementioned process, the enzyme can beco-deposited with a polymeric membrane by exposing the working electrodeto a solution containing both the enzyme and a monomer and by applying aconstant potential or current between the working and the referenceelectrode. A constant potential is preferred for this enzyme deposition.The concentration of the monomer can be varied and the monomer can beselected from the group consisting of ortho-phenylene diamine (OPD),para-phenylene diamine, meta-phenylene diamine, pyrrole, flavins,naphthalene, aniline, phenols, thiophenes, sulfonated aniline,sulfonated pyrrole, and the like. In an exemplary embodiment, themonomer is OPD.

In a variation, the enzyme is first deposited on the wire that forms theworking electrode before disposing it in the conduit.

In one embodiment, the enhanced activity of the decorated nanostructuredmaterial is preserved by periodic cleaning of the working electrode in abuffer solution, by any one of processes selected from electrochemicalcleaning, etching, sonication, or combinations thereof. Electrochemicalcleaning is preferred.

In a variation of the aforementioned methodology, the cleaning isperformed in a buffer solution whose pH is acidic, neutral or alkaline.In an exemplary embodiment, the cleaning is performed in an acidicbuffer solution. In a variation of the aforementioned methodology, thecleaning is performed in a quiescent, stirred or flowing buffer solutionand preferably in a flowing buffer solution.

In another embodiment, the cleaning is performed in a non-quiescentenvironment by desorbing the poisoning species andadsorbing/polymerizing them onto a nearby electrically conductivestructure. Such a structure can be an overlayer on top of thepitted/etched and nanomaterial-decorated working electrode edge. Thisoverlayer can comprise a polymeric film made up of but not limited topolyvinylchloride, polycarbonate, polyvinylacetate, humic acids,cellulose acetate, polythiophenes, polyphenylene diamines, polypyrroles,polynaphthalenes, polyphenols, or the like, or a combination thereof. Inaddition, this over layer can be deposited via spin coating, dropcasting, dip coating, layer-by-layer assembly, inkjet printing, spraycoating and electropolymerization. In an exemplary embodiment, theoverlayer is deposited via electropolymerization.

The sensor works on the principle of electrochemical oxidation orreduction of the electroactive species that is either present in the invivo environment or produced as a result of reaction between the enzymeand the analyte to be detected. The electrochemical oxidation orreduction is achieved by applying a potential between the workingelectrode wire and reference electrode wire. This electrochemicaloxidation or reduction produces a current at the working electrode thatcan be measured using an electronic unit.

This sensor is advantageous in that it is suitable for the simultaneousdetection of more than one analyte. This can be readily accomplished byincreasing the number of wires in the conduit or the number of metalconducting lines in the tape based ‘edge’ biosensors. The tape comprisesa material selected from the group consisting of paper, polyester,polyimide, polyetherimide, polyolefin, polytetrafluoroethylene,polysiloxane, or a combination of at least one of the foregoingmaterials.

In the event that additional wires are disposed in the conduit, eachwire is coated with an enzyme specific to the analyte of interest. Allother processes such as pitting/etching, decoration with nanostructuredmaterials and cleaning remain the same.

In the event that additional wires are disposed in the conduit, eachwire can be independently addressed via an active matrix element. TheFIG. 10 is a depiction of an exemplary sensor that comprises a pluralityof wires (electrodes) that are greater in number than the 3 electrodesof the FIGS. 1A and 2D. With respect to the FIG. 10, the sensorcomprises an array of wires (electrodes) 1002 disposed in anelectrically insulating casing 1004. The insulating casing 1004comprises an internal cavity termed the sensor housing 1008. The sensorhousing has an external surface 1010 that forms the outside of thesensor housing 1010. The electrodes 1002 are disposed on an activematrix addressing chip 1006. The active matrix addressing chip 1006along with the electrodes are disposed in the cavity on the inner sideof the sensor housing 1008. A cross-sectional surface area of theelectrodes are exposed to contact analytes for purpose of detection andanalysis.

In one embodiment, a method for manufacturing the sensor of the FIG. 10comprises disposing an array of thin wires (electrodes) disposed withina conduit where the thin wires are electrically separated by aninsulating layer. The conduit is then cut to expose edges (crosssectional area) of the electrodes. The exposed edges areelectrochemically etched to increase their surface area. Theelectrochemically etched surface is coated with nano-sizedelectro-catalytic moieties suitable for the catalysis of a given redoxspecies, where the electrocatalytic moieties are surface coated with athin polymeric film that is semi permeable to small molecules butimpermeable to large molecular interferences. The nano-sizedelectro-catalytic moieties can be regenerated by applying a cyclicpotential in an aqueous solution. The nano-sized electro-catalyticmoieties when contacted by the metabolite can produce an electricalsignal that is detected with the appropriate microelectronics.

The opposing cross sectional surface area of the electrodes is inelectrical communication with an active matrix that in turn communicateswith appropriate microelectronics for signal detection. The activematrix is capable of switching and interrogating between differentelectrodes.

In order to expose a cross-sectional surface of the electrodes, afterthe wires are disposed in the conduit, both sides of the conduit are cutperpendicular to the long axis of the conduit to expose the wire edges.On one side, all processes such as pitting/etching, decoration withnanostructured materials and cleaning are performed. The other side ofthe edges are connected to an active matrix (via flip-chip bonding,ultra sonic bonding, electropolymerization, conductive adhesives,soldering micro- or nanobeads, and the like) that is in turn connectedto the appropriate microelectronics for signal detection. The activematrix is capable of switching and interrogating between different saidthin wires and minimizes the number of interconnects within the deviceencasing.

This sensor is advantageous in that it allows for the elimination of nonspecific adsorption of one enzyme on another working electrode duringthe fabrication of the aforementioned multi-analyte sensors. This isachieved by masking the concerned working electrodes via dipping theelectrode in electroplating solutions (such as Cu, Cr, Ag, Zn, and thelike), thiol or allyl derivatives solution, which can be removedsubsequently by applying a fixed potential.

Another advantage of this sensor is its ability to perform an internalcalibration routine to account for foreign body response and biofouling,which are known to degrade enzyme activity and analyte flux. This can beachieved by utilizing two working electrodes, wherein both the surfacesare pitted/etched and decorated with the nanostructured materials, butonly one is coated with an enzyme. This enzyme is selected in such a waythat it specifically reacts with the analyte of interest to produce aproduct which is electroactive at the surface of the working electrode.The internal calibration of the enzyme coated working electrode can beachieved by comparing its background currents with that of thebackground currents of the blank working electrode and through thedifferential offsetting of the background currents of the enzymaticworking electrodes with respect to the blank working electrode. Sincethe background current is independent of enzymatic activity and analyteflux, the procedure will allow for an estimation of the magnitude of theforeign body response and therefore will facilitate internal selfcalibration.

In yet another advantage, the sensor is suitable to achievethree-dimensional diffusion of the analyte towards the workingelectrode. Three-dimensional diffusion of analyte towards the workingelectrode is preferred in order to improve sensor sensitivity and limitof detection. The sensor of FIG. 1A (100) or FIG. 2 (200) by itselfaffords three dimensional diffusion, but it can be further enhanced byincreasing the number of wires in the conduit or the number of metalconducting lines in the tape based ‘edge’ biosensors. All otherprocesses such as pitting/etching, decoration with nanostructuredmaterials and cleaning remain the same.

The invention will be illustrated in more detail with reference to thefollowing examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

This example was conducted to demonstrate a method for pitting/etchingon the surface of the working electrode to enhance its electrochemicalarea. The pitting/etching of the surface of the working electrode isachieved by exposing the working electrode to a stirred solution of 2 MNaOH (sodium hydroxide) solution and by applying a sequential potentialof +0.8 V (volt) and −1 V for more than 15 minutes. As can be seen inFIG. 3, the response current increases with increasing time which is anindication of the pitting/etching process that also results in anincreased surface area of the of the working electrode (see FIG. 1B).

Example 2

This example was conducted to demonstrate a method of increasing theelectrochemical activity of the working electrode by sequentialprocesses involving the pitting/etching of the surface of the workingelectrode followed by decoration with nanostructured materials.

At first the sensor of FIG. 1A is exposed to a stirred solution of 2MNaOH, while applying a sequential potential of +0.4 V and −0.8 V forabout 15 minutes. Subsequently, this surface is decorated with platinumnanoparticles, by exposing the surface to 10 mM H₂PtCl₆ (chloroplatinicacid hexahydrate) in 0.1 M HCl and simultaneously applying a constantcurrent of 12 microamperes/square millimeter for 15 minutes.

FIG. 4 illustrates the sensitivity of the sensor to H₂O₂ when tested at0.6 V with respect to Ag/AgCl reference. As can be seen in FIG. 4, thepitting/etching of the surface of the working electrode enhances the(hydrogen peroxide) H₂O₂ sensitivity by 2 orders of magnitude, owing tothe increased electrochemical active area (FIG. 1B). This H₂O₂sensitivity is further enhanced by an additional 25% by decorating thepitted/etched surface of the working electrode with a thin layer ofplatinum nanoparticles. (FIG. 1C)

Example 3

This example illustrates a methodology to improve sensor sensitivity bya simple electrochemical cleaning process. The biosensor of FIG. 1A isdecorated with platinum nanoparticles, as described in Example 2 and itssensitivity towards H₂O₂ before and after electrochemical cleaning isinvestigated. The electrochemical cleaning step involves subjecting theworking electrodes to cyclic voltammetric sweeps (21 cycles in range−0.5 to +0.9 V at a rate of 100 millivolts/sec) in flowing phosphatebuffer saline (PBS, pH=7.4) (30 microliters/minute).

FIG. 5 shows the amperometric current versus H₂O₂ concentration for the“as fabricated” biosensor when operated at 0.4 V versus Ag/AgCl. Theslopes of these curves represent the sensitivity for H₂O₂ detection. Thesensitivity of the freshly deposited Platinum-black electrodes increasedfrom 0.092 nA·μM⁻¹ mm⁻² of the bare gold electrode to 0.65 nA·μM⁻¹ mm⁻².Following electrochemical cleaning, the sensitivity of the sensortowards H₂O₂ increased by 33 times versus that of freshly depositedplatinum electrodes, to a value of 22 nA·μM⁻¹ mm⁻². This value is240-fold larger than that for the Au electrodes alone. This improvementmay be due to flow-induced removal of various adsorbed residues on theplatinum black surfaces, along with the selective oxidation of platinumatoms that promote the formation of selected crystallographic planes.

Example 4

This example demonstrates the microelectrode behavior of edge planesensors of the present disclosure. For this, the sensor of FIG. 1A(before and after subjecting to electrochemical etching and subsequentdeposition of platinum nanoparticles) was subjected to cyclicvoltammetry (CV) in 10 mM K₃(FeCN₆) in 1M KCl aqueous solution at 10mV/s scanning rate and the corresponding CV curves are shown in FIG. 6.The CV curves of these sensors exhibited a sigmoidal shape which istypical of microelectrode behavior. Moreover, the sigmoidal shape wasretained even after electrochemical etching and platinum nanoparticledecoration of the working electrode.

Example 5

This example, illustrates a methodology wherein the biosensor of FIG. 1Acan be decorated with a nanostructured material to enhance sensitivity,via by co-depositing the nanostructured material with a polymericmembrane. Apart from the enhancement in the sensor sensitivity, thismethodology affords a significant advantage of sensor selectivityagainst electro-active endogenous species such as ascorbic acid, uricacid, acetaminophen, and the like. The latter is typically achieved viathe use of ultra-thin electropolymerized films that act as permselectivemembranes that inadvertently reduces sensor sensitivity towards H₂O₂.The biosensor of FIG. 1 is coated with a thin layer ofelectropolymerized film of poly (phenylene diamine) (PPD) in thepresence and absence of nanostructured materials based on flavin-wrappedsingle walled carbon nanotubes (FMN-NT). FIG. 7A shows the amperometricresponse of PPD and PPD+FMN-NT coated working electrodes to sequentialadditions of H₂O₂ operated at 0.7 V versus Ag/AgCl reference. As can beseen, both the sensors displayed an increase in their response uponaddition of H₂O₂. However, for all H₂O₂ additions, the PPD+FMN-NT actedsensors showed a higher response compared to PPD coated sensors.

FIG. 7B illustrates the response of the PPD and PPD+FMN-NT coated sensoras a function of H₂O₂ glucose concentration. For both the sensors, theaddition of H₂O₂ resulted in a linear increase in their amperometricresponse within the range of H₂O₂ tested. The PPD+FMN-NT sensors,however displayed a 5-6 fold higher response compared to PPD sensors,owing the enhanced electroactivity of the FMN-NTs.

Example 6

This example illustrates a methodology to obtain stablemicro-electrodes. The stable microelectrodes are obtained on pre-cleanedglass slides. These substrates were then spin coated with 1.2 mm ofphotoresist film followed by baking (120° C., 10 min.) Following UVexposure, development and plasma cleaning, a 10 nm (nanometer)chromium/100 nm gold layer was evaporated and lifted off (in acetone) toafford electrode patterning. Subsequently, following an 8 hour vacuumtreatment in hexamethyldisilazane (HMDS) vapors, a 2 μm thick positivephotoresist was deposited and patterned to create coarse openings overall electrodes and contact pads. 1 hour of exposure in a gold platingsolution (10 μA·mm⁻² current density) enabled the electroplating of 5 μmthick Au over the exposed gold electrodes. Subsequently, the entiredevice was rinsed in acetone to remove all photoresist, and re-spuncoated with 2 μm of a final positive photoresist to define the finalelectroactive area of electrodes (with a circular opening of 900 μm indiameter) and 2×5 mm rectangular windows for contact pads. Workingelectrodes were realized by electrodepositing 300 nm of platinum in 10mM H₂PtCl₆ and 0.1 M HCl at 12 μA·mm⁻² for 15 minutes. The finalstructure of working electrode is shown in FIG. 8.

Example 7

This example illustrates a methodology to modify a particular workingelectrode (without affecting the other working electrodes) of amicroelectrode array containing a plurality of more than 3 workingelectrodes. This methodology is illustrated with a sensor similar to theone shown in FIG. 1A containing three working electrodes, one referenceand one counter electrode. The sensor to be fabricated will be utilizedas a multi-analyte device for simultaneous detection of glucose, lactateand oxygen. The detection of glucose and lactate is achieved bymeasuring the amperometric current produced by electrochemical oxidationof H₂O₂, produced as result of enzymatic reaction of glucose and lactatewith glucose oxidase and lactate oxidase, respectively. Oxygen detectionis achieved by measuring the amperometric current produced by itselectrochemical reduction.

The methodology to immobilize the specific enzyme on the specificworking electrode involves a masking technique based on electrochemicaldeposition of copper and its subsequent removal via electrochemistry inneutral, aqueous solutions. A detailed schematic of this technique isshown in FIG. 9A. FIG. 9B shows the in vitro operation of the asfabricated multi-analyte sensor in a stirred PBS buffer solutions. Thesensor was operated by biasing the glucose and lactate working electrodeat 0.7 versus Ag/AgCl reference and the oxygen working element at −0.1 Vversus Ag/AgCl reference. As seen in FIG. 9B, the addition of glucose tothe test cell resulted in an increase in the response of only theglucose working element. Similarly, the addition of lactate to the testcell resulted in an increase in the response of only the lactate workingelement. Here it is worth noting that the response of the oxygen sensingelement remained constant throughout the course of the experiment. Thesefacts are indicative of the zero chemical crosstalk among these sensorswhich in turn indicate that the masking technology demonstrated in thisexample is highly efficient to modify only one and only one workingelectrode.

Example 8

This example illustrates a schematic to produce an array of highlysensitive microelectrode devices that are independently addressed via anactive matrix element. This device is composed of array of thin wiresembedded at one edge within a flexible encasing and are electricallyseparated from each other by an insulating layer. Such array can beproduced by fusing multiple insulated wires, where the insulation hasfilled all the gaps. Alternatively, glass coated wires are fusedtogether or co-extruded to form arrays of desired size and density.These are then cut into a thin slice and polished on both sides toexpose the edges of the said micron wires. One of the exposed edges areelectrochemically etched to increase their surface area and subsequentlycoated with nano-sized electro-catalytic moieties suitable for thecatalysis of a given redox species. The other exposed edges areconnected to an active matrix (via flip-chip bonding, ultra sonicbonding, electropolymerization, conductive adhesives, soldering micro-or nanobeads, and the like) that is in turn connected to an appropriatemicroelectronics for signal detection. The active matrix is capable ofswitching and interrogating between different said thin wires andminimizes the number of interconnects within the device encasing.

The sensors disclosed herein can be advantageously used for thesimultaneous and independent determination of metabolites in the body ofa living being. These sensors are highly flexible and are miniaturized.The have a high electro-active area and display 3-dimensional analytediffusion. They afford high enzyme loadings and permit high sensorperformance and usability. They can be fabricated in a manner thatpermits them being rolled up for storage. They can be manufactured in alarge scale roll-to-roll process. The biosensor is also capable ofdetecting analytes with a low applied potential. They display very lowdetection limits and display no interferences from endogenous species.They have a high signal to noise ratio.

In one embodiment, the sensor has a diameter of about 10 micrometers toabout 1,000 micrometers, specifically about 20 micrometers to about 500micrometers, and more specifically about 30 micrometers to about 250micrometers. Having such a narrow diameter (or thickness in the case ofthe “tape” sensor) allows the shape of the sensor to be adjusted andreadjusted if desired. In one embodiment, the sensor is flexible enoughthat its shape can be adjusted by hand without the use of any tools.

While the invention has been described with reference to someembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing fromessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiments disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

What is claimed is:
 1. A method comprising: disposing a plurality ofwires within a conduit; the wires being electrically separated by aninsulating layer; cutting the conduit into a cut conduit having a firstside and an opposite second side; polishing the first and second sidesof the cut conduit to expose a first cross-sectional area and a secondcross-sectional area of the plurality of the wires at the first side andthe second side of the cut conduit respectively; electrodepositing asacrificial layer on the exposed first cross-sectional area of theplurality of the wires; as step #1, selecting wires andelectrochemically etching the sacrificial layer on the selected wire toexpose a cross-sectional surface of the selected wire; as step #2,electrochemically etching the exposed cross-sectional surface of theselected wire to produce nanostructure porosity; as step #3, coating theexposed cross-sectional surface of the selected wire having thenanostructure porosity with an electro-catalytic moiety suitable forcatalyzing a redox reaction of a given redox species to form a workingelectrode; as step #4, performing an electrochemical cleaning process toclean the working electrode; as step #5, electrodepositing a film of apermselective membrane to an exposed cross-sectional surface area of theworking electrode; as step #6, depositing first enzymes, which reactswith a first analyte to produce an electroactive species, onto thepermselective membrane to render the working electrode, together with areference electrode and a counter electrodes operative to detect thefirst analytes via an electrochemical oxidation or reduction reaction;selecting a second wire from the plurality of the wires and performingsteps #1 to #6 to form a second working electrode having a second enzymedeposited on a second permselective membrane of the second workingelectrode; selecting a third wire from the plurality of the wires andperforming steps #1 to #4 or steps #1 to #5 to form a referenceelectrode; connecting the second cross-sectional area of the pluralityof the wires to an active matrix, wherein the active matrix iselectrically connected to microelectronic device for signal detection;and wherein the active matrix is operative to control which wire orwires among the plurality of the wires are used in detecting signals. 2.The method of claim 1, wherein each of the plurality of the wires iscoated with an electro-catalytic moiety composed of platinumnanoparticles.
 3. The method of claim 1, wherein the electrodepositedsacrificial layer is deposited via an electrochemical process.
 4. Themethod of claim 3, wherein the electrodeposited sacrificial layercomprises copper.
 5. The method of claim 1, wherein the electrodepositedsacrificial layer is removed via an electrochemical process.
 6. Themethod of claim 1, wherein the electro-catalytic moiety suitable forcatalyzing the redox reaction of the given redox species is depositedvia an electrochemical process.
 7. The method of claim 1, wherein thepermselective membrane is deposited via an electrochemical process. 8.The method of claim 7, wherein the said-permselective membrane comprisesan electropolymerized flavin mononucleotide.
 9. The method of claim 7,wherein the said-permselective membrane comprises an electropolymerizedmixture of flavin mononucleotide and nanotubes.
 10. The method of claim7, wherein the permselective membrane comprises an electropolymerizedmixture of flavin mononucleotide, nanotubes and conductive polymersformed from at least one monomer of ortho-phenylene diamine (OPD),para-phenylene-diamine, meta-phenylene diamine, phenol, pyrrole,flavins, naphthalene, polyaniline, aniline, thiophenes, sulfonatedaniline, or sulfonated pyrrole.
 11. The method of claim 1, wherein theelectro-catalytic moiety is specific to a metabolite.
 12. The method ofclaim 10, wherein the electro-catalytic moiety comprises glucoseoxidase.
 13. The method of claim 1, wherein each of the plurality of thewires has a diameter of about 5 nanometers to about 1000 micrometers.